Establishment and application of a quadruple real-time RT-PCR for detecting avian metapneumovirus

In order to develop an appropriate method for high-throughput detection of avian metapneumovirus, a quadruple real-time reverse-transcription polymerase chain reaction assay was established with four pairs of specific primers and four specific probes based on the G or M gene of aMPV-A, aMPV-B, aMPV-C and aMPV-D. Its specificity and sensitivity were evaluated, and clinical samples were tested by the method. The results showed that all the four subgroups of avian metapneumovirus can be detected in the quadruple real-time RT-PCR assay simultaneously, with a detection limit of 100–1000 cRNA copies/reaction. The other common poultry viruses were negative. In the avian clinical sample detection, 39 out of 1920 clinical samples collected from 8 provinces were positive. Compared with published RT-PCR assays, the κ value of the quadruple real-time RT-PCR assay in 1920 avian clinical samples was 1.000 (P < 0.001). The established method could be used for the rapid detection of the four subgroups of avian metapneumovirus with high specificity and high sensitivity.


Introduction
Avian metapneumovirus (aMPV) has been detected for a long time [1]. aMPV can cause both respiratory and reproductive disorders in a variety of poultry, including turkeys, chickens, guinea fowls and pheasants [2]. Four subgroups exist based on the antigenic and molecular variations, termed aMPV-A, aMPV-B, aMPV-C and aMPV-D [3][4][5]. aMPV was first discovered in South Africa in 1970, spread throughout Europe in the 1980s and was found in the United States in 1996. In recent years, the detection of aMPV has been reported in many countries or regions, especially in Europe [6][7][8][9][10][11][12].
aMPV was found in China in 1999, but the subgroup was not identified. Subsequently, many researchers carried out a series of surveys on chicken infection with aMPV. In 2007 and 2008, serological tests were carried out on breeder flocks in some areas of Shandong province, and the results showed that the seropositive rate of several flocks reached 100% [13]. In 2015, 296 serum samples were collected from 9 chicken farms in China, and the observed aMPV seropositivity rate was 74.3% [14]. Multiple survey data indicated that aMPV has become prevalent among chickens in China. However, only aMPV-C was isolated from broilers in southeastern China [10] and Muscovy ducks [15]. aMPV-B was isolated from chickens in Liaoning province [16], whereas aMPV-A was detected using reverse transcription-polymerase chain reaction (RT-PCR) [17]. aMPV-D was not detected in China. The detection methods of aMPV include virus isolation, enzyme-linked immunosorbent assay (ELISA) and RT-PCR [18][19][20]. Virus isolation is not generally used as a routine detection for aMPV because of its difficult. aMPV could not be subtyped using Some established ELISA methods. Moreover, these methods are time-consuming and insensitive, and are not suitable for rapid detection of aMPV. Some real-time RT-PCR methods have also been established which could not detected all subtypes of aMPV [21]. In order to improve the detection methods of aMPV and clarify the impact of aMPV on poultry in China, a quadruple real-time RT-PCR was established in this study, which can detect all four subgroups of aMPV simultaneously. The method was evaluated using 1920 avian clinical samples from 8 provinces of China.

Ethics approval and consent to participate
This study was conducted according to the animal welfare guidelines of the World Organization for Animal Health and approved by the Animal Welfare Committee of the China Animal Health and Epidemiology Center. The approval number was DWFL202104.

Sample collection and RNA extraction
The aMPV-B positive sample was donated by Zhejiang Agriculture & Forestry University, and the aMPV-C positive sample was donated by the Shanghai Animal Disease Prevention and Control Center. Avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis (ILTV) were all conserved in the China Animal Health and Epidemiology Center. The 1920 cloacal/throat double swabs were obtained from 25 live poultry markets or farms in 8 provinces of China according to a random sampling method. Live poultry markets or farms of 8 provinces in China were selected at random in 2019. The 1920 samples were collected from five animal species, including white pigeon, goose, chicken, duck, and partridge. The samples were stored in 0.5 mL phosphate-buffered saline (PBS, pH 7.2) containing 10% glycerol at 4˚C to 8˚C. Viral RNA was extracted within 3 days after collection. The remaining samples were stored at −80˚C. The samples were centrifuged at 12,000 g at 4˚C for 5 min. Viral RNA was extracted according to the manufacturer's instructions of the cador Pathogen 96 QIAcube HT kit (Qiagen, Hilden, Germany) and stored at −80˚C.

Synthesize RNA in vitro
The four standard plasmids were digested by enzyme (SpeⅠand NotⅠ) and purified with the DNA Extraction Kit (Solarbio, Beijing, China) respectively. The purified DNA was used as template for in vitro transcription reactions with RiboMAX TM Large Scale RNA Production System-SP6 and T7 kit (Promega, USA). The mixture of transcription reaction in vitro include 20 μL of 5× T7 Transcription Buffer, 7.5 μL of rNTPs (25mM ATP, CTP, GTP, UTP), 10 μL T7 Enzyme mix, 30 μL Linear DNA template and 32.5μL Nuclease Free H 2 O. The transcription program was 37˚C for 5 h. In order to remove the DNA template, 10 μL RQ1 RNase-Free DNase was added to the reaction system and incubated at 37˚C for 15 min. Then, the miscellaneous proteins and various ions in the system were removed with the Rneasy MiNi Kit (Qiagen, Hilden, Germany), and the cRNA was extracted and purified. The cRNA was quantified using a Thermo Scientific Multiskan GO Microplate Photometer (Thermo Fisher Scientific, USA). The RNA copy number was calculated according to the following formula: RNA copy number per μL = (6.02 × 10 23 × plasmid concentration (ng/μL) × 10 −9 )/(RNA length × 340).

aMPV real-time RT-PCR primers and probe design
aMPV-A, aMPV-B and aMPV-D specific primers and probes were designed based on the G gene of aMPV. The M gene was selected as reference sequence for aMPV-C for primer and probe design. A total of 36 available G gene sequences of aMPV-A, 36 available G gene sequences of aMPV-B, 40 available M gene sequences of aMPV-C and 3 available G gene sequences of aMPV-D from NCBI GenBank database were aligned to check the specific and conserved regions with Molecular Evolutionary Genetics Analysis (MEGA) software 7.0 respectivly. Multiple primers and probes were designed for each subgroup. The design and evaluation of these primers and probes were performed using Oligo7 software [22]. The primers and probes specific to each subgroup of aMPV were grouped and tested to select the optimal primers and probes. When screening primers and probes, the risk of cross-reactivity issues for different subgroups should be avoided. The Taq-Man hydrolysis probes were labeled with fluorescence reporter dye (ROX, FAM, HEX, and CY5) at the 5 0 -end and quenched with BHQ1 or BHQ2 at the 3 0 -end. Details of the final primers and exo-probes are shown in Table 1. All primers and exo-probes were synthesized by Sangon.

Condition optimization
The reaction condition of the quadruple real-time RT-PCR was optimized, including the concentration of primers and probes, temperature and time of annealing/extension. All primers and probes were dissolved to 10 pmol/μL. The optimum reaction concentration of primers and probes were selected by different volumes (list in S2 Table). Five annealing/extension temperature (58˚C, 59˚C, 60˚C, 61˚C, 62˚C) and three annealing/extension time (20s, 30s, 40s) were tested to screen the best annealing/extension conditions.

Analytical sensitivity of the quadruple real-time RT-PCR
The analytical sensitivity of each component in the quadruple real-time RT-PCR was evaluated using a tenfold serial dilution of standard recombinant plasmids for the four subgroups (10 6 -10 1 cRNA copies per reaction). Evaluation of the analytical sensitivity of the quadruple real-time RT-PCR method was performed by detecting each dilution in eight replicates.

Analytical specificity of the quadruple real-time RT-PCR
The specificity of the quadruple real-time RT-PCR for aMPV was evaluated using one aMPV-B, one aMPV-C, 12 AIVs, one NDV, 1 ILTV and two IBVs. The details of all the virus are listed in Table 2.

Evaluation of the quadruple real-time RT-PCR assay using avian clinical sample
In total, 1920 avian double swabs were detected and compared with the published RT-PCR assay for the four subgroups of aMPVs to evaluate the performance of the quadruple real-time RT-PCR assay. The primers for the published RT-PCR assay [18] are listed in Table 1. According to the published methods, RT-PCR for the four subgroups of aMPVs was performed using the Prime-Script ™ One-step RT-PCR Kit Ver. 2 (Takara, Dalian, China; RR055A), and the reaction system included 12.5 μL of 2× One-step buffer, 1 μL of each primer (10 pM), 1 μL of PrimeScript 1 Step Enzyme Mix, 3 μL of nucleic acids, and 6.5 μL of RNase-free H 2 O. The reaction conditions were as follows: 30 min at 50˚C, 4 min at 95˚C and 40 cycles of 98˚C for 30 s, 58˚C for 30 s and 72˚C for 30 s, followed by a final extension step for 7 min at 72˚C. Then the amplified DNA fragments were sequenced using synthetic oligonucleotides (Sangon Biotech, Shanghai, China). To verify the positive results by conventional PCR, the sequences of the PCR products were compared with the GenBank nucleotide database by online BLAST search at the NCBI website.

Quadruple real-time RT-PCR
The quadruple real-time RT-PCR reaction system was performed according to the optimal conditions and programs. The results of the optimal conditions and programs were showed in S3-S6 Tables. The reaction

Sensitivity of the quadruple real-time RT-PCR assay
The sequences of the quadruple real-time RT-PCR primers and TaqMan probes are listed in Table 1. The aMPV-A, aMPV-B and aMPV-D molecular plasmid standards were positive in the assays from 10 3 RNA copies per reaction. Six, four and four of eight replicates with 10 2 RNA copies per reaction tested positive in the quadruple real-time RT-PCR assay. For aMPV-C, reactions were positive from 10 4 copies per reaction. Five of eight replicates with 10 3 RNA copies per reaction tested positive in the quadruple real-time RT-PCR assay. The detection limits of the quadruple real-time RT-PCR assay at 95% were 10 2 RNA copies per reaction for aMPV-A, aMPV-B and aMPV-D and 10 3 RNA copies per reaction for aMPV-C (probit analysis, P < 0.05). The detection results of the aMPV-A, aMPV-B, aMPV-C and aMPV-D standards are shown in Table 3 and Fig 1, respectively.

Specificity of the quadruple real-time RT-PCR
The quadruple real-time RT-PCR assay was positive for the plasmid of four aMPV subgroups and negative for the samples, including AIVs, NDVs, IBVs and negative controls. aMPV-B

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and aMPV-C positive samples also tested positive in the quadruple real-time RT-PCR assay, but they tested negative to the probes for aMPV-A and aMPV-D. Thus, the quadruple realtime RT-PCR assay demonstrated high specificity for the detection of all subgroups of aMPV (Fig 2).

Evaluation of the real-time RT-PCR assay using avian clinical samples
The real-time RT-PCR assay detected 39 positive samples out of 1920, in agreement with the results of the RT-PCR assay. The sensitivity of the RT-PCR was tested by the four standard plasmids. The κ value for RT-PCR and real-time RT-PCR was 1.000 (P < 0.001) ( Table 4).

Epidemiological analysis of aMPV
Out of 1920 avian clinical samples, 39 (2.03%) were positive. The geography and host distribution of the 39 positive samples were analyzed. Whenever one or more sample from a live Table 3.   Table 6.

Discussion
aMPV-A, aMPV-B and aMPV-C have been detected in China with various methods [16,17,23]. aMPV-D has never been found in China. aMPV vaccine has not been used in China. In order to prevent spreading of the disease and facilitate treatment, early detection of viral infection, especially the establishment of a rapid and accurate detection method, has received much attention. Most real-time PCR detection technologies employ the TaqMan fluorescent probe method. Compared with the traditional SYBR Green method, this method is highly specific. In this

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study, an aMPV quadruple real-time RT-PCR detection method was established using Taq-Man fluorescent probes, which can quickly and accurately detect aMPVs and can be used for large-scale detection of aMPV infection in poultry flocks. Subtyping of aMPV was based on G gene. The G gene sequences of all aMPV subgroups were downloaded from NCBI. Through homology comparison, it was found that the G gene sequence of aMPV-C was poorly conserved, so the more conserved M protein gene sequence was used as a template to design primers and probes for aMPV-C detection. The G genes of aMPV-A, aMPV-B and aMPV-C are more conserved, and can be used to design detection primers and probes. Therefore, the quadruple real-time RT-PCR detection method was established based on the G genes of aMPV-A, aMPV-B and aMPV-D and the M genes of aMPV-C.

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The aMPV detection method was developed based on quadruple real-time RT-PCR, while genotyping was performed simultaneously. Compared with RT-PCR and virus isolation, it can reduce the probability of contamination, it can yield experimental results in a short time and it has higher specificity and sensitivity. The nucleic acid samples of aMPV-B and aMPV-C were positive in the quadruple real-time RT-PCR assay. All aMPV positive samples and four standard plasmids used for the evaluation of the quadruple real-time RT-PCR assay were positive. Due to the limitation of the source of aMPV-A and aMPV-D in China, it is currently impossible to test the ability of this method to detect these two subgroups with positive virus samples. However, according to the primer/exo-probe design method and the plasmid standard detection results, the quadruple real-time RT-PCR assay is considered to be suitable for the detection of aMPV-A and aMPV-D.
In order to evaluate the quadruple real-time RT-PCR assay and investigate the prevalence of aMPV in China, 1920 double swabs of live poultry trading markets in eight provinces of Shanghai, Anhui, Jiangxi, Jiangsu, Hubei, Hebei, Guangxi and Guangdong were collected. A total of 39 samples were found to be positive, including 32 aMPV-B and seven aMPV-C samples. The clinical samples tested positive by the quadruple real-time RT-PCR were also confirmed to be positive by PCR detection and sequencing. The test results showed that 28 aMPV-B positive samples were from chicken and four aMPV-B positive samples were from duck. Moreover, six duck samples and one goose sample tested positive for aMPV-C. It can be seen that aMPV-B is common in chickens in China, and aMPV-C is common in ducks. aMPV-C is often reported in geese abroad [24][25][26]. In our study, aMPV-C in geese was discovered for the first time in China. According to the distribution of the aMPV positive rate, Jiangxi province occupies 9.2%, Shanghai 3.3%, Jiangsu and Guangxi 1.3% and Anhui, Hubei and Guangdong provinces each 0.4%, and aMPV was not detected in Hebei province. This clinical sample test proves that most aMPV infection in chickens in China are subgroup B, and a small number of infected ducks and goose have subgroup C. Effective measures should be taken to prevent spreading of the disease and avoid economic losses.

Conclusion
The quadruple real-time RT-PCR was established in this study can detect all four subgroups of aMPV simultaneously, is not time-consuming and has good specificity and high sensitivity. This assay provides a powerful tool for the detection of aMPVs, which is important for the control of aMPV.
Supporting information S1 Table. The sensitivity test results of the RT-PCR method for aMPV. The plasmid of aMPV-A, aMPV-B, aMPV-C and aMPV-D were diluted series for the sensitivity tests. The number of copies per reaction was counted which could be tested positive. The minimum number of copies per reaction was considered to the sensitivity for the four subgroup of aMPV. (DOCX) S2 Table. The 81 combinations of primers and probes with different volume in the reaction mixture. Table. The test results of different primers and probes volume in the reaction mixture. The quadruple real-time RT-PCR reaction system was performed according to the BioScript (Dalian) Co., Ltd. PrimeScriptTM One Step RT-PCR Kit instructions. The standard recombinant plasmids for the four subgroups (10 4 cRNA copies per reaction) were used as templates. The mean values of CT were calculated to analyze and screen the optimal volume of primers and probes. (DOCX) S4 Table. The mean values of CT. The results showed that the mean values of CT were decreasing with the increasing of primer and probes volumes. However, when the volume of primers and probes increased from 1.0μL to 1.4μL, the CT values dose not decreased significantly. Therefore, considering the cost and results of the experiment, the volume of primers and probes for the four subgroups were set as 1.0μL in the 50μL reaction mixture. The reaction mixture contained the following: 25.0 μL of 2× One Step RT-PCR Buffer Ⅲ, 1.0 μL of 5 U/μL TaKaRa Ex Taq Table. The test results of optimum annealing/extension temperature for the quadruple real-time RT-PCR. Five annealing/extension temperature (58˚C, 59˚C, 60˚C, 61˚C, 62˚C) were tested to screen the optimal annealing/extension conditions. The results showed that CT values were smaller and the sensitivity was higher than other temperatures when the annealing/extension temperature was 59˚C or 60˚C. In the appropriate temperature range, the specificity of primers and probes will increase with the raising of annealing/extension temperature. Therefore, the annealing/extension temperature of the quadruple real-time RT-PCR was set as 60˚C.
(DOCX) S6 Table. The results of optimum annealing/extension time for the quadruple real-time RT-PCR. Three annealing/extension time (20s, 30s, 40s) were tested to screen the optimal annealing/extension conditions. The results showed that there was no significant difference in the CT values when the annealing/extension time was 20s, 30s and 40s respectively. The annealing/extension time was set as 30s to prevent the influence of stability of the experiment by too short annealing/extension time. The quadruple real-time RT-PCR cycler conditions were as follows: 30 min at 50˚C, 4 min at 95˚C and 40 cycles of 98˚C for 30 s, 60˚C for 30 s and 72˚C for 30 s, followed by a final extension step for 7 min at 72˚C.